Related Technical Notes

Click-iT Reagents for Detecting Protein Synthesis and Modifications

Click-iT labeling technology employs a bioorthogonal reactive chemistry for the in situ labeling of specific molecular populations, such as proteins that have been newly synthesized or post-translationally modified in some experimental time window of interest. The Click-iT labeling reaction is based on a copper-catalyzed azide–alkyne cyloaddition and derives its high degree of specificity from the fact that the azide and alkyne reaction partners have no endogenous representation in biological molecules, cells, tissues or model organisms.

Application of this reaction to in situ labeling of cells is a two-step process. First, one reaction partner—either an azide or alkyne linked to a "building block" such as an amino acid, monosaccharide, fatty acid, nucleotide or nucleoside—is biosynthetically incorporated. Subsequently, the other reaction partner—the complementary alkyne or azide linked to a fluorescent dye, biotin or other detection reagent—is "clicked" into place in the presence of catalytic copper (I), providing a detection moiety (Figure 9.4.1). One reaction partner must be an azide derivative and the other an alkyne derivative, but either functional moiety can serve as the biosynthetically incorporated molecule or the detection molecule (e.g., L-azidohomoalanine (AHA) + Alexa Fluor 488 alkyne is the inverse of the reaction scheme shown in Figure 9.4.1A).

The small size of alkyne and azide tags allows the biosynthetic building blocks to which they are attached to be processed by enzymes, such as aminoacyl tRNA synthetases and nucleotide polymerases, that have poor tolerance for substrates with larger modifications such as fluorescent organic dyes. Furthermore, the 1,2,3-triazole linkage between the azide and alkyne reaction partners (Figure 9.4.1) is extremely stable. It is not susceptible to hydrolysis, oxidation or reduction, and it survives ionization in mass spectrometry (MS) analysis. Click-iT labeling technology and the details of the click reaction are discussed in Click Chemistry—Section 3.1. For a complete list of azide and alkyne derivatives compatible with Click-iT labeling technology, see Molecular Probes azide and alkyne derivatives—Table 3.1, Click Chemistry—Section 3.1. Here we highlight the azide and alkyne derivatives that can be used for labeling newly synthesized proteins and detecting post-translational protein modifications.

Figure 9.4.1 Click-iT copper-catalyzed azide–alkyne cycloaddition chemistry applied to detection of A) proteins and B) carbohydrates. The reaction partners are A) L-homopropargylglycine (HPG) and Alexa Fluor 488 azide and B) N-azidoacetylgalactosamine and Alexa Fluor 488 alkyne. In both cases, the left-hand partner is a metabolic precursor that can be incorporated into proteins via de novo synthesis or post-translational modification pathways.

Azide- and Alkyne-Modified Amino Acids

The Click-iT AHA (L-azidohomoalanine, C10102) and Click-iT HPG (L-homopropargylglycine, C10186; Figure 9.4.1A) reagents are methionine surrogates that provide nonradioactive alternatives to 35S-methionine for pulse-chase detection of protein synthesis and degradation. These amino acid analogs are fed to cultured cells and incorporated into proteins during active protein synthesis. The enzymatically incorporated Click-iT AHA or Click-iT HPG is then detected with a fluorescent alkyne or fluorescent azide, respectively, using a Cu(I)-catalyzed click reaction. These Click-iT reagents provide detection sensitivity comparable to that obtained using the radioactive 35S-methionine method and are compatible with downstream LC-MS/MS and MALDI MS analysis, as well as with total-protein, glycoprotein and phosphoprotein gel stains for differential analyses of newly synthesized protein together with post-translational modifications.

Click-iT AHA is also available in the Click-iT AHA Alexa Fluor 488 Protein Synthesis HCS Assay Kit (C10289), which provides Alexa Fluor 488 alkyne for detection. Click-iT AHA has proven to be a successful substitute for methionine in many cell types, including COS-7, 3T3-L1, HeLa, HEK 293 and Jurkat cells. Note that cells should be labeled in methionine-free media, as methionine is the preferred substrate for methionyl tRNA transferase, and supplemented media (i.e., methionine-free DMEM) should be used in place of HBSS to achieve greater Click-iT AHA incorporation at lower concentrations.

Azide- and Alkyne-Modified Monosaccharides

The Click-iT metabolic glycoprotein labeling reagents provide biosynthetic precursors for detecting and characterizing post-translational glycosylation of proteins. Four azide- or alkyne-modified monosaccharides are available for metabolic incorporation into a specific subclass of protein glycan structures:

Cultured cells are simply incubated with the modified sugars for 2–3 days or until cells reach the appropriate density. The acetyl groups improve cell permeability of the modified sugars and are removed by nonspecific intracellular esterases (Figure 9.4.2). The resulting azide- or alkyne-modified sugar is then metabolically incorporated through the permissive nature of the oligosaccharide biosynthesis pathway, yielding functionalized glycoproteins that can be chemoselectively coupled to complementary alkyne- or azide-functionalized fluorophores and biotinylation reagents for detection or affinity capture. We offer three Click-iT Protein Analysis Detection Kits (C33370, C33371, C33372) described below for the detection of azide-functionalized glycoproteins in 1D or 2D electrophoresis gels or western blots. These labeled glycoproteins are compatible with total-protein, glycoprotein and phosphoprotein gel stains and provide a detection sensitivity of a few hundred femtomoles, allowing an in-depth analysis of low-abundance glycoproteins as well as glycoproteins with a small degree of glycosylation.

Glycoproteins labeled with the Click-iT labeling and detection reagents are also compatible with downstream LC-MS/MS and MALDI-MS analyses for further identification and characterization. For added convenience, we offer an O-GlcNAc peptide LC/MS standard (C33374) from the transcription factor CREB for LC-MS/MS and MALDI-MS analyses of the O-GlcNAc posttranslational modification. This peptide is also available together with its phosphorylated counterpart for use as LC/MS standards (C33373) in differential mass spectrometry–based studies of the corresponding modifications, as well as for characterizing differential β-elimination/addition conditions.

Figure 9.4.2 Metabolic incorporation of tetraacetylated azido sugars.

Click-iT O-GlcNAc Enzymatic Labeling System

We also offer the the Click-iT O-GlcNAc Enzymatic Labeling System for in vitro enzyme-mediated N-azidoacetylgalactosamine labeling of O-GlcNAc–modified glycoproteins (C33368). Proteins are enzymatically labeled using the permissive mutant β-1,4-galactosyltransferase (Gal-T1, Y289L), which transfers azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAc residues on the target proteins. Target proteins can then be detected using an alkyne-derivatized fluorophore or one of the Click-iT Protein Analysis Detection Kits described below. Using the Click-iT O-GlcNAc Enzymatic Labeling System in conjunction with the Click-iT Tetramethylrhodamine (TAMRA) Protein Analysis Detection Kit, we have detected as little as 1 picomole of α-crystallin, a protein which is only 2–10% O-GlcNAc modified.

Pro-Q Diamond Phosphoprotein Stain for Gels and Blots

We have developed a suite of compatible methodologies for the differential staining of specific proteins (phosphoproteins, glycoproteins or membrane proteins) and the total-protein profile in two or more visually distinguishable colors, producing a more complete picture of the proteome. This set of protein stains not only offer the capacity for the simultaneous detection of multiple protein targets in a single sample, but also provides a combination of high sensitivity and simplicity that can streamline protocols in 1D and 2D polyacrylamide gels or on blots.

The Pro-Q Diamond phosphoprotein gel stain, Pro-Q Emerald glycoprotein gel stains and SYPRO Ruby protein gel stain—which we have optimized to complement each other in selectivity, sensitivity and staining protocols—can be used in serial detection of phosphoproteins, glycoproteins and total proteins on a single protein sample separated by 1D or 2D gel electrophoresis (Figure 9.4.3). Our Rhinohide polyacrylamide gel strengthener (R33400, Protein Detection on Gels, Blots and Arrays—Section 9.3) greatly improves the strength of any polyacrylamide gel, making it easy to perform these multiple staining procedures without special gel handling. After each staining step, an image of the gel is collected. Once collected, the three images can be overlaid in any combination for analysis of phosphorylation, glycosylation and total-protein expression. Because all three stains are used on the same gel, unambiguous spot matching of phosphoproteins and glycoproteins is made simple by direct comparison with the total-protein profile provided by the SYPRO Ruby protein gel stain. This simultaneous measurement of several variables ensures perfect spatial registration of signals and increases the amount of data that can be collected in a single experiment, leading to more controlled experiments, more accurate data comparisons and fewer ambiguities.

Figure 9.4.3 An overview of the Multiplexed Proteomics approach. Images collected after each staining step can be overlaid in any combination for analysis of protein expression, phosphorylation and glycosylation between samples.

Pro-Q Diamond Phosphoprotein Gel Stain and Destain

Pro-Q Diamond phosphoprotein gel stain is a breakthrough technology that provides a simple, direct method for selectively staining O-linked phosphoproteins in polyacrylamide gels (Figure 9.4.4). It is ideal for the identification of kinase targets in signal transduction pathways and for phosphoproteomic studies. This proprietary fluorescent stain allows direct, in-gel detection of phosphate groups attached to tyrosine, serine or threonine residues. The Pro-Q Diamond phosphoprotein gel stain can be used with standard SDS-polyacrylamide gels (Figure 9.4.4) or with 2D gels (). The simple and reliable staining protocol delivers results in as little as 4 to 5 hours. The stain is also compatible with mass spectrometry, allowing analysis of the phosphorylation state of entire proteomes. The Pro-Q Diamond phosphoprotein gel stain provides:

Simple in-gel detection. Proteins containing phosphate groups attached to tyrosine, serine or threonine residues can be detected directly in either 1D or 2D polyacrylamide gels after the gel is fixed, stained and destained; no antibodies are required and no blotting is necessary.

Selectivity without radioactivity. The Pro-Q Diamond phosphoprotein gel stain is a fluorescent stain that selectively detects phosphoproteins; radioisotopes are not used and therefore no radioactive waste is generated.

Sensitivity. The Pro-Q Diamond phosphoprotein gel stain allows the detection of as little as 1–16 ng of phosphoprotein per band, depending on the phosphorylation level of the protein.

Quantitation. The Pro-Q Diamond signal for individual phosphoproteins is linear over three orders of magnitude and correlates with the number of phosphate groups (Figure 9.4.5).

Multiplexing capability. Pro-Q Diamond gel stain can be used with SYPRO Ruby protein gel stain (Figure 9.4.4) and Pro-Q Emerald glycoprotein gel stain on the same gel for multiparameter staining.

The Pro-Q Diamond phosphoprotein gel stain (Pro-Q Diamond gel stain reagents and kits—Table 9.4) is supplied ready-to-use in three different sizes: a 200 mL size (P33301) suitable for staining approximately four minigels; a 1 L size (P33300) suitable for staining approximately 20 minigels or two large-format gels, e.g., 2D gels; and a 5 L bulk-packaging size (P33302). In addition, we offer Pro-Q Diamond Phosphoprotein Gel Staining Kits (MPP33300, MPP33301, MPP33302) that include both the Pro-Q Diamond gel stain and the PeppermintStick phosphoprotein molecular weight standards (see below). All products are accompanied by a simple and reliable staining and destaining protocol that delivers results in as little as four to five hours. For convenient destaining, we also offer the Pro-Q Diamond phosphoprotein gel destaining solution as a ready-to-use solution in either a 1 L (P33310) or 5 L (P33311) size.

Figure 9.4.4 Selectivity of the Pro-Q Diamond phosphoprotein gel stain (P33300, P33301, P33302) for phosphoproteins. A polyacrylamide gel containing various proteins was stained with A) Pro-Q Diamond phosphoprotein gel stain, followed by B) SYPRO Ruby protein gel stain. This gel shows a nonphosphorylated protein, lysozyme (lanes 3 and 4), as well as several phosphoproteins, α-casein (lanes 1 and 2), ovalbumin (lanes 5 and 6) and pepsin (lanes 7 and 8), before (even lanes) and after (odd lanes) treatment with phosphatases. Loss of Pro-Q Diamond staining indicates loss of all phosphates from pepsin, partial loss of phosphates from α-casein and ovalbumin and no change in the nonphosphorylated protein lysozyme.

Figure 9.4.5 Sensitivity and linear range of Pro-Q Diamond phosphoprotein gel stain (P33300, P33301, P33302). Six different proteins were serially diluted and run on separate SDS-polyacrylamide gels, which were then stained with Pro-Q Diamond phosphoprotein gel stain. The images were documented on a fluorescence imager, and the fluorescence emission from each band was quantitated. The number of known phosphate groups on each protein is indicated in the figure legend. A) Fluorescence emission of the band, plotted as a function of protein amount, in nanograms. B) Magnification of data points in the highlighted box in panel A. C) The fluorescence emission of the band, plotted as a function of picomoles of protein. D) Magnification of the data points in the highlighted box in panel C. E) The slope of the line for each protein in panel C, plotted against the known number of phosphates per protein.

When used together, the Pro-Q Diamond phosphoprotein gel stain and the SYPRO Ruby protein gel stain (S12000, S12001, S21900; Protein Detection on Gels, Blots and Arrays—Section 9.3) make a powerful combination for proteome analysis. The SYPRO Ruby dye is a total-protein stain that, like the Pro-Q Diamond gel stain, is quantitative over three orders of magnitude. Determining the ratio of the Pro-Q Diamond dye to SYPRO Ruby dye signal intensities for each band or spot thus provides a measure of the phosphorylation level normalized to the total amount of protein. Using both stains in combination makes it possible to distinguish a low amount of a highly phosphorylated protein from a higher amount of a less phosphorylated protein. To make this staining more convenient and economical, we offer the Multiplexed Proteomics Kit #2 with 200 mL of the Pro-Q Diamond phosphoprotein gel stain and 200 mL of the SYPRO Ruby protein gel stain (M33306), the Multiplexed Proteomics Kit #1 with 1 L of each stain (M33305) and the Multiplexed Proteomics Phosphoprotein Gel Stain Kits (MPM33305, MPM33306), which include the Pro-Q Diamond phosphoprotein gel stain, SYPRO Ruby protein gel stain and PeppermintStick phosphoprotein molecular weight standards (see below); Pro-Q Diamond gel stain reagents and kits—Table 9.4 summarizes all of our Pro-Q Diamond gel stain reagents and kits.

Pro-Q Diamond Phosphoprotein Blot Stain Kit

The Pro-Q Diamond Phosphoprotein Blot Stain Kit (P33356) provides a simple and quick method for directly detecting phosphoproteins on poly(vinylidene difluoride) (PVDF) or nitrocellulose membranes without the use of radioactivity or antibodies. As with the gel stain, the Pro-Q Diamond phosphoprotein blot stain detects phosphoserine-, phosphothreonine- and phosphotyrosine-containing proteins, independent of the sequence context of the phosphorylated amino acid residue. Thus, the native phosphorylation levels of proteins from a variety of sources, including tissue specimens and body fluids, can be analyzed. Protein samples are separated by 1D or 2D gel electrophoresis, electroblotted to the membrane, stained and destained using a protocol similar to that typically performed with amido black or Ponceau S staining of total-protein profiles on membranes. After staining, gels are simply imaged using any of a variety of laser scanners, xenon-arc lamp–based scanners or CCD-based imaging devices employing UV transilluminators; the excitation/emission maxima of the Pro-Q Diamond phosphoprotein blot stain are ~555/580 nm (Figure 9.4.6). The limits of detection for the stain on PVDF membrane blots are typically 8–16 ng of phosphoprotein, with a linear dynamic range of approximately 15-fold. The sensitivity of the Pro-Q Diamond phosphoprotein blot stain is decreased when using nitrocellulose blots. Each Pro-Q Diamond Phosphoprotein Blot Stain Kit provides sufficient reagents for staining ~20 minigel electroblots, including:

The Pro-Q Diamond phosphoprotein blot stain binds noncovalently to phosphoproteins and is thus fully compatible with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and Edman sequencing. Furthermore, the Pro-Q Diamond phosphoprotein blot stain is compatible with the standard colorimetric, fluorometric and chemiluminescent detection techniques employed in immunoblotting. This phosphoprotein blot stain may be used in conjunction with the SYPRO Ruby protein blot stain, a total-protein stain that is quantitative over two orders of magnitude on blots. Using the SYPRO and Pro-Q Diamond blot stains in combination makes it possible to distinguish a low amount of a highly phosphorylated protein from a higher amount of a less phosphorylated protein.

PeppermintStick Phosphoprotein Molecular Weight Standards

PeppermintStick phosphoprotein molecular weight standards are a mixture of phosphorylated and nonphosphorylated proteins with molecular weights from 14,400 to 116,250 daltons. Separation by polyacrylamide gel electrophoresis resolves this mixture into two phosphorylated and four nonphosphorylated protein bands (). These standards serve both as molecular weight markers and as positive and negative controls for our Pro-Q Diamond phosphoprotein gel stain and other methods that detect phosphorylated proteins. We offer two different unit sizes of the PeppermintStick phosphoprotein molecular weight standards: a 40 µL unit size sufficient for 20–40 gel lanes (P27167) and a 400 µL unit size sufficient for 200–400 gel lanes (P33350).

Phosphopeptide Standard Mixture

Formulated especially for MALDI-MS, the phosphopeptide standard mixture (P33357) contains equimolar amounts of three unphosphorylated and four phosphorylated peptides, ranging in mass between 1047 and 2192 and representing phosphoserine (pS), phosphothreonine (pT) and phosphotyrosine (pY) monophosphopeptides, as well as a peptide containing both pT and pY. This mixture is ideal for use as an internal or external control for LC/MS, MALDI analysis or β-elimination reactions.

Pro-Q Diamond Phosphoprotein Enrichment Kits

The Pro-Q Diamond Phosphoprotein Enrichment Kit (P33358) enables efficient, nonradioactive isolation of phosphoproteins from complex cellular extracts. This kit provides resin, reagents and columns designed to isolate phosphoproteins from 0.5–1.0 mg of total cellular protein per column. The column bed volume can be easily scaled up or down depending on the amount of available starting material. The phosphoprotein-binding properties of the resin allow efficient capture of both native and denatured proteins. Therefore, cell or tissue samples can be denatured in lysis buffers and stored in the freezer prior to the phosphoprotein enrichment procedure. Each Pro-Q Diamond Phosphoprotein Enrichment Kit contains:

Protocols for both undenatured and denatured lysates are provided, and these procedures can be completed in approximately three hours. For added convenience, the Pro-Q Diamond Phosphoprotein Enrichment and Detection Kit (P33359) provides all the reagents in the Pro-Q Diamond Phosphoprotein Enrichment Kit, as well as Pro-Q Diamond phosphoprotein gel stain and PeppermintStick phosphoprotein molecular weight markers for detecting phosphoproteins on SDS-polyacrylamide gels.

Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit

The Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit (P33706) provides a method for selective staining of phosphoproteins or phosphopeptides on microarrays, without the use of antibodies or radioactivity. This kit permits direct detection of phosphate groups attached to tyrosine, serine or threonine residues in a microarray environment and has been optimized for microarrays with acrylamide gel surfaces. Each Pro-Q Diamond Phosphoprotein/Phosphopeptide Microarray Stain Kit provides:

Sufficient reagents are provided for 20 HPLC separations; a single separation will selectively detect 20 picomoles or less of a monophosphorylated peptide using a standard microbore C18 HPLC column.

Other Reagents for Phosphoproteomics

The Antibody Beacon Tyrosine Kinase Assay Kit (A35725), described in detail in Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3, provides a simple yet robust solution assay for real-time monitoring of tyrosine kinase activity and the effectiveness of potential inhibitors and modulators. The key to this tyrosine kinase assay is a small-molecule tracer ligand labeled with our bright green-fluorescent Oregon Green 488 dye. When an anti-phosphotyrosine antibody binds this tracer ligand to form the Antibody Beacon detection complex, the fluorescence of the Oregon Green 488 dye is efficiently quenched. In the presence of a phosphotyrosine-containing peptide, however, this Antibody Beacon detection complex is rapidly disrupted, releasing the tracer ligand and relieving its antibody-induced quenching. Upon its displacement by a phosphotyrosine residue, the Oregon Green 488 dye–labeled tracer ligand exhibits an approximately fourfold enhancement in its fluorescence, enabling the detection of as little as 50 nM phosphotyrosine-containing peptide with excellent signal-to-background discrimination.

Pro-Q Glycoprotein Stain Kits for Gels and Blots

Glycoproteins play important roles as cell-surface markers, as well as in cell adhesion, immune recognition and inflammation reactions. To facilitate research on glycoproteins, we offer the Pro-Q Glycoprotein Stain Kits for Gels and for Blots, which provide extraordinary sensitivity, linearity and ease of use for selective detection of glycoproteins.

Pro-Q Emerald Glycoprotein Stain Kits for Gels and for Blots

Pro-Q Emerald 300 and Pro-Q Emerald 488 Glycoprotein Stain Kits (Pro-Q Emerald glycoprotein stain kits for gels and for blots—Table 9.5) provide advanced reagents for detecting glycoproteins in gels and on blots. The Pro-Q Emerald glycoprotein stains react with periodate-oxidized carbohydrate groups, creating a bright green-fluorescent signal on glycoproteins (Figure 9.4.7). The staining procedure requires only three steps: fixation, oxidation and staining—no reduction step is required. Depending on the nature and degree of glycosylation, the Pro-Q Emerald 300 stain allows the detection of as little as 1 ng of a glycoprotein per band in gels (4 ng/band with the Pro-Q Emerald 488 stain), making these stains about 50 times more sensitive than the standard periodic acid–Schiff base method using acidic fuchsin dye. Blot staining is not quite as sensitive (2–18 ng of a glycoprotein per band can be detected) and is more time consuming, but provides an opportunity to combine glycoprotein staining with immunostaining or other blot-based detection techniques. The Pro-Q Emerald 300 stain is best visualized using 300 nm UV illumination, whereas the Pro-Q Emerald 488 stain is best visualized using visible light with wavelengths near its 510 nm excitation maximum. The Pro-Q Emerald dye is also used as the detection reagent in our Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495), which is described in Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3.

Figure 9.4.7 Detecting glycoproteins with Pro-Q Emerald glycoprotein detection reagents. Oxidation with periodic acid converts cis-glycols to dialdehydes, which can then react with the hydrazide-based Pro-Q Emerald reagents to form a covalent bond.

The Pro-Q Emerald glycoprotein stains can be combined with general protein stains for dichromatic detection of glycoproteins and total proteins in gels and on blots, making it much easier to identify the location of the glycoproteins in the total-protein profile (Figure 9.4.8, Figure 9.4.9, Figure 9.4.10, ). The SYPRO Ruby protein gel and blot stains (described in Protein Detection on Gels, Blots and Arrays—Section 9.3) provide the same sensitivity as silver staining (gels) or colloidal gold staining (blots) but, unlike these chromogenic techniques, do not require formaldehyde or glutaraldehyde, which can produce false-positive responses when glycoproteins are stained. These total-protein stains make it possible to visualize the entire protein complement of a sample and to thus identify contaminating proteins, to compare stained proteins to molecular weight standards and to provide a control for protease contamination in glycosidase mobility-shift experiments. The SYPRO Ruby protein blot stain is additionally useful for assessing the efficiency of protein transfer to a blot (Figure 9.4.10), which is especially important when working with glycoproteins, because they often transfer poorly to blotting membranes. Proteins labeled with the SYPRO Ruby total-protein stains exhibit red-orange fluorescence when excited with either a 300 nm UV light source or a laser scanner with a 473, 488 or 532 nm laser light source.

Figure 9.4.9 Mobility-shift gel assay using deglycosylating enzymes, stained with the SYPRO Ruby protein gel stain (top; S12000, S12001, S21900) and Pro-Q Emerald 300 glycoprotein stain (bottom). The glycoproteins α1-acidic glycoprotein, fetuin and horseradish peroxidase (HRP) are shown before (lanes 2, 4 and 6, respectively) and after (lanes 3, 5 and 7, respectively) glycosidase treatment. Glycosidase treatment resulted in a mobility shift and loss of green-fluorescent Pro-Q Emerald 300 staining for α1-acidic glycoprotein and fetuin, indicating that the carbohydrate groups had been cleaved off. HRP, which contains an α-(1,3)-fucosylated asparagine GlcNac-linkage that is resistant to many glycosidases, showed neither a mobility shift nor a loss of green-fluorescent Pro-Q Emerald 300 staining. Thus, use of the Pro-Q Emerald 300 Glycoprotein Stain Kits (P21855, P21857, M33307) identifies glycoproteins that are not susceptible to the glycosidases used in the assay, providing important structural information about the glycoprotein's carbohydrate moiety.

Figure 9.4.10 Staining glycoproteins and the total protein profile on blots using the Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain Kit (P21857). A twofold dilution series of the CandyCane glycoprotein molecular weight standards (C21852) was run an SDS-polyacrylamide gel and blotted onto a PVDF membrane. The blot was first stained with the SYPRO Ruby protein blot stain (S11791) to detect the total protein profile (left). After documentation of the signal, the blot was stained with the Pro-Q Emerald 300 glycoprotein stain (right) provided in the Pro-Q Emerald 300 Glycoprotein Gel and Blot Stain Kit.

When used together, the Pro-Q Emerald 300 glycoprotein gel stain and the SYPRO Ruby protein gel stain (S12000, S12001, S21900; Protein Detection on Gels, Blots and Arrays—Section 9.3) make a powerful combination for proteome analysis (Figure 9.4.8). Determining the ratio of the Pro-Q Emerald dye to SYPRO Ruby dye signal intensities for each band or spot provides a measure of the glycosylation level normalized to the total amount of protein. Using both stains in combination makes it possible to distinguish a lightly glycosylated, high-abundance protein from a heavily glycosylated, low-abundance protein. To make this staining more convenient and economical, we offer the Multiplexed Proteomics Glycoprotein Gel Stain Kit with 1 L of our Pro-Q Emerald 300 glycoprotein gel stain and 1 L of our SYPRO Ruby protein gel stain (M33307).

CandyCane Glycoprotein Molecular Weight Standards

CandyCane glycoprotein molecular weight standards (C21852) contain a mixture of glycosylated and nonglycosylated proteins with molecular weights from 14,000 to 180,000 daltons). When separated by polyacrylamide gel electrophoresis, the standards appear as alternating bands corresponding to glycosylated and nonglycosylated proteins (Figure 9.4.11). Thus, these standards serve both as molecular weight markers and as positive and negative controls for methods that detect glycosylated proteins, such as those provided in our Pro-Q Emerald Glycoprotein Stain Kits (see above).

Figure 9.4.11 Glycosylated and nonglycosylated proteins in the CandyCane glycoprotein molecular weight standards (C21852). The standards were electrophoresed through two identical 13% polyacrylamide gels. Both lanes contain ~0.5 µg of protein in each band. The left lane was stained with our SYPRO Ruby protein gel stain (S12000, S12001, S21900) to detect all eight marker proteins. The right lane was stained using the reagents in the Pro-Q Emerald 300 Glycoprotein Gel Stain Kit (P21855).

Other Specialized Techniques for Detecting Specific Proteins on Gels and Blots

Detecting Calcium-Binding Proteins

The luminescent lanthanide terbium, which is available as its chloride salt (Tb3+ from TbCl3, T1247), selectively stains calcium-binding proteins in SDS-polyacrylamide gels. With some modifications to the staining protocol, these lanthanides can also be used to detect all protein bands. Terbium chloride has also been used as a rapid negative stain for proteins in SDS-polyacrylamide gels, in which the background is green fluorescent and the proteins are unstained.

Detecting Penicillin-Binding Proteins

BOCILLIN FL penicillin and BOCILLIN 650/665 penicillin (B13233, B13234) are green- and infrared-fluorescent penicillin analogs, respectively, that bind selectively and with high affinity to penicillin-binding proteins present on the cytoplasmic membranes of eubacteria. When electrophoresed under nonreducing conditions, the dye-labeled penicillin-binding proteins are easily visible in the gel with sensitivity in the low nanogram range (Figure 9.4.12). BOCILLIN FL penicillin, synthesized from penicillin V and the BODIPY FL dye (spectrally similar to fluorescein), has been used to determine the penicillin-binding protein profiles of Escherichia coli, Pseudomonas aeruginosa and Streptococcus pneumoniae, and these binding profiles are found to be similar to those reported by researchers using radioactively labeled penicillin V. Fluorescently labeled penicillin has also been used for direct labeling and rapid detection of whole E. coli and Bacillus licheniformis and of Enterobacter pneumoniae. The β-lactam sensor-transducer (BlaR), an integral membrane protein from Staphylococcus aureus, covalently and stoichiometrically reacts with β-lactam antibiotics, including BOCILLIN FL penicillin, by acylation of its active-site serine residue.

Figure 9.4.12 Detection of penicillin-binding proteins (PBPs) from Escherichia coli and Pseudomonas aeruginosa. The membrane fractions from E. coli and P. aeruginosa were prepared as previously described and labeled with BOCILLIN 650/665 penicillin (B13234). The labeled membranes were separated on an SDS-polyacrylamide gel, stained with SYPRO Ruby and visualized using a Typhoon imager (Molecular Dynamics). The location of PBPs from E. coli are labeled to the left of the gels. Lanes 1, 3, and 5 are E. coli membrane preparations; lanes 2, 4, and 6 are P. aeruginosa membrane preparations; lanes 1 and 2 are overlays of images obtained from total protein (green) and PBP (red) scans; lanes 3 and 4 are total protein visualized with the SYPRO Ruby protein gel stain; lanes 5 and 6 are PBPs as detected by BOCILLIN 650/665. Image used with permission from Wiley VCH publishers.

TC-FlAsH and TC-ReAsH detection technology, based on the tetracysteine tag first described by Griffin, Adams and Tsien in 1998, takes advantage of the high-affinity interaction of a biarsenical ligand (FlAsH-EDT2 or ReAsH-EDT2) with the thiols in a tetracysteine (TC) expression tag fused to the protein of interest. The FlAsH-EDT2 ligand is essentially fluorescein that has been modified to contain two arsenic atoms at a set distance from each other, whereas the ReAsH-EDT2 ligand is a similarly modified resorufin. Virtually nonfluorescent in the ethanedithiol (EDT)-bound state, these reagents become highly fluorescent when bound to the thiol-containing tetracysteine tag Cys-Cys-Xxx-Yyy-Cys-Cys, where Xxx-Yyy is typically Pro-Gly (Figure 9.4.13). Modified tags with additional flanking sequences produce higher affinity binding of the biarsenical ligand, resulting in improved signal-to-background characteristics. Selective labeling of two proteins for fluorescence microscopy colocalization and FRET analysis has been accomplished using TC tags with different binding affinities in combination with FlAsH-EDT2 and ReAsH-EDT2.

Transfecting the host cell line with an expression construct comprising the protein of interest fused to a tetracysteine tag (CCPGCC) is the first step in TC-FlAsH TC-ReAsH detection. The tagged protein is then detected by the addition of FlAsH-EDT2 reagent (Figure 9.4.13) or ReAsH-EDT2 reagent, generating green or red fluorescence, respectively, upon binding the tetracysteine motif. For detection of tetracysteine-tagged proteins expressed in cells, we offer the TC-FlAsH II and TC-ReAsH II In-Cell Tetracysteine Tag Detection Kits (T34561, T34562, T34563), which are described in Thiol-Reactive Probes Excited with Visible Light—Section 2.2.

Figure 9.4.14 Protein gel staining using TC-FlAsH Expression Analysis Detection Kit (A10068). A) Tetracysteine-tagged proteins are labeled with FlAsH-EDT2 reagent and fluoresce green. B) Total proteins are labeled with the Red total-protein stain provided in the kit and fluoresce red. C) An overlay of the two images reveals relative amounts of protein.

Detecting Oligohistidine Fusion Proteins

The oligohistidine domain is a Ni2+-binding peptide sequence comprising a string of four to six histidine residues. When the DNA sequence corresponding to the oligohistidine domain is fused in frame with a gene of interest, the resulting recombinant protein can be easily purified using a nickel-chelating resin.

Developed by QIAGEN, the Penta·His mouse IgG1 monoclonal antibody (P21315) provides a sensitive method for specific detection of fusion proteins that have an oligohistidine domain comprising five or six consecutive histidine residues. The antibody does not recognize tetrahistidine domains or domains in which the histidine string is interrupted by another amino acid. The Penta·His antibody binds to the oligohistidine domain regardless of the surrounding amino acid context and even when the group is partially hidden, although subtle differences in the amino acid context may change the sensitivity limit for a particular fusion protein. The antibody is ideal for detecting oligohistidine fusion proteins on western blots (). The Penta·His antibody is also useful for immunoprecipitation, ELISA assays and immunohistochemistry.

In protein fusion techniques, the coding sequence of one protein is fused in-frame with another so that the expressed hybrid protein possesses desirable properties of both parent proteins. One common partner in these engineered products is glutathione S-transferase (GST), a protein with natural binding specificity that can be exploited to facilitate its purification. Because the GST portion of the fusion protein retains its affinity and selectivity for glutathione, the fusion protein can be conveniently purified from the cell lysate in a single step by affinity chromatography on glutathione agarose (Figure 9.4.16). For purification of GST fusion proteins, we offer glutathione linked via the sulfur atom to crosslinked beaded agarose (10 mL of sedimented bead suspension, G2879). Each milliliter of gel can bind approximately 5–6 mg of bovine-liver GST. Adding excess free glutathione liberates the GST fragment from the matrix, which can then be regenerated by washing with a high-salt buffer.

We also offer a highly purified rabbit polyclonal anti-GST antibody (A5800) that can be used to purify GST fusion proteins by immunoprecipitation. This highly specific antibody, which was generated against a 260–amino acid N-terminal fragment of the Schistosoma japonica enzyme expressed in Escherichia coli, is also useful for detecting GST fusion proteins on western blots and for detecting GST distribution in cells. The intensely green-fluorescent Alexa Fluor 488 conjugate of anti–glutathione S-transferase (A11131) is also available for direct detection of GST fusion proteins.

Following purification, the fusion protein can serve as an immunogen for antibody production or its properties can be compared with those of the native polypeptide to provide insights on the normal function of the polypeptide of interest. Such methods have been used to investigate biological properties of many proteins. Examples include cleavage of the capsid assembly protein ICP35 by the herpes simplex virus type 1 protease, the role of the Rho GTP-binding protein in lbc oncogene function and the association of v-Src with cortactin in Rous sarcoma virus–transformed cells. In fact, the Ca2+-binding properties of a protein kinase C–GST fusion protein were examined while the GST fusion protein was still bound to the glutathione agarose. Likewise, interactions of a DNA-binding protein–GST fusion protein have been assessed using an affinity column consisting of the fusion protein bound to glutathione agarose. Alternatively, the GST fusion expression vector can be engineered to encode a recognition sequence for a site-specific protease, such as thrombin or factor Xa, between the GST structural gene and gene of interest. Once the fusion protein is bound to the affinity matrix, the site-specific enzyme can be added to release the protein.

Streptavidin Acrylamide and Reactive Acrylamide Derivatives

Streptavidin acrylamide (S21379), which is prepared from the succinimidyl ester of 6-((acryloyl)amino)hexanoic acid (acryloyl-X, SE, A20770), may be useful for the preparation of biosensors. A similar streptavidin acrylamide has been shown to copolymerize with acrylamide on a polymeric surface to create a uniform monolayer of the immobilized protein. The streptavidin can then bind biotinylated ligands, including biotinylated hybridization probes, enzymes, antibodies and drugs.

Like streptavidin and CaptAvidin biotin-binding protein, other amine-containing biomolecules can be crosslinked to acrylamides using acryloyl-X, SE. Acryloyl-X, SE reacts with amines of proteins, amine-modified nucleic acids and other biomolecules to yield acrylamides that can be copolymerized into polyacrylamide matrices or on surfaces, such as in microarrays and in biosensors. We prepare both streptavidin and CaptAvidin biotin-binding protein conjugated to 4% beaded crosslinked agarose (S951, C21386; Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6)—matrices that can be used to isolate biotinylated peptides, proteins, hybridization probes, haptens and other molecules.